CONCLUSION Atmospheric pressure ionization (MI)mass spectrometry is a new instrumental method which provides femtogram detection capabilities and selective ionization conditions for many compounds of biologic and medical interest. Perhaps the most remarkable feature is that for some purposes extracts of biologic material may be injected directly. It now seems possible to develop direct LC-MS-COM systems by directing liquid chromatography effluents into the vaporization zone, and to develop GC-MS-COM sys-
tems based upon high resolution (more than 100,000 theoretical plates) thermostable glass capillary columns (7, 8). Received for review July 23, 1973. Accepted December 6, 1973. This work was aided by Grants GM-13901 and GM16216 of the National Institute of General Medical Sciences, Contract NIH 69-2161 of the National Institute of General Medical Sciences, Grant HE-05435 of the National Heart and Lung Institute, and Grant Q-125 of the Robert A. Welch Foundation. (7) A. L. German and E. C. Horning. J . Chromatogr. Sa., 11, 76 (1973). (8) A. L. German, C. ,D. Pfaffenberger, J-P. Thenot, M. G. Horning, and E. C. Horning. Anal. Chem., 45, 930 (1973).
Direct Determination of Heavy Elements in Biological Media by Spark Source Mass Spectrometry A. W. Fitchett' and R. P. Buck William Rand Kenan, J r . , Laboratories of Chemistry, University of North Carolina, Chapel Hill, N.C. 275 74
Paul Mushak Department of Pathology, University of North Carolina, Chapel Hill, N.C. 27574
The analysis of trace heavy elements in biological media by spark source mass spectrometry has been studied. The direct analysis of serum, homogenized liver, and urine is accomplished without prior ashing. It has been found that by proper control of the sparking parameters, organic interferences can be held to the lower mass range, thus leaving the higher range free from such interfering ions. The elements studied were Pt, Au, Hg, TI, Pb, and Bi. Through the use of an internal image developer and elemental calibration curves, concentrations below 1 ppm have been determined from single exposures. Unique sampling procedures and feasibility as a routine pathological tool have been discussed.
The identification and study of trace heavy metals in biological systems is of ever increasing importance, particularly in the areas of clinical and forensic pathology as well as environmental toxicology. Of special interest in this regard are bioanalytical methodologies which permit the simultaneous evaluation of a number of elements, in many cases it being more desirable to assess level ratios of elements rather than isolated values. Currently, there exist a number of multiple element procedures with major use made of neutron activation analysis ( I ) , X-ray fluorescence ( 2 ) ,emission spectroscopy ( 3 ) ,and spark-source mass spectrometry ( 4 ) . Activation analysis is valuable where sensitivity is an overriding criterion but is a procedure requiring highly sophisticated instrumentation and manipulation, rendering the technique relatively inaccessible to the general analytical community. Emission spectroscopy, a method enjoyPresent address, D e p a r t m e n t of Pathology, N o r t h Carolina, C h a p e l Hill, N.C. 27514.
University
of
( 1j G. H . Morrison and N. M Potter, Anal. Chem., 44, 839 (1972). (2) K Martin, Musz. Tud.. 44, 363 (1971) (3) W . Slavin. "Emission Spectrochemical Analysis," Wiley-lnterscience, New York, N Y., 1971 ( 4 ) A J Ahearn. Ed., "Trace Analysis by Mass Spectrometry,'' Academic Press, New York, N . Y . , 1972.
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ing perhaps longest use, classically yields poorest precision among the various methods. X-Ray fluorescence spectrometry, in many cases, does not possess sufficient sensitivity without recourse to sample concentrating steps. Spark-source mass spectrometry (SSMS) with its capability for low detection limits, minimal sample requirements, and moderately accessible instrumentation, renders it increasingly useful for multiple-element bioassay in various media. This report concerns itself with results of the analysis of selected biological substrates for multiple element levels using spark-source mass spectrometry and direct sampling of the media studied. The use of SSMS in analysis of biological samples is not a new subject (5-11). Evans and Morrison (9) have evaluated the applicability of SSMS for the quantitative determination of trace elements in biological materials, but their study did not emphasize the heavy element region or a direct sampling procedure. In most pertinent reports to date, samples received pretreatment including ashing, wet or dry (high or low temperature). In wet ashing, the introduction of contaminants is a major problem while in high or low temperature ashing, sample loss due to volatilization is of acute concern. Owing to these drawbacks, direct sampling was studied in great detail. This study demonstrates that SSMS analysis may be achieved with a variety of biological media without prior ashing. The selected elements under investigation were Pt, Au, Hg, T1, Pb, and Bi in lyophilized blood serum, (5) N. Sasaki and E. Watanabe, Thirteen Ann. Conf. Mass Specfrom. Allied Topics, 473 (1965) (6) R Brown, et ai., Fifteenth Ann. Conf. Mass Specfrom. Allied Topics. 157 (1967). (7) S. Klugeand H . J. Dietze, Z. Naturforsch.. 236, 1393 (1968) (8) R . M. Jones, W. F Kuhn. and C Varsel, Anal. Chem., 40, 10 (1968). (9) C. A. Evansand G . H. Morrison, Anal. Chem., 40,869 (1968) (10) J. P Yurachek, G G Clemena, and W . W. Harrison, Anal. Chem.. 41, 1666 (1969). (11) S. S. C. Tong, W. H. Gutenmann, and D. J. Lisk, Anal. Chem.. 41, 1872 (1969)
homogenized liver, and urine. The latter four elements are of extreme importance in that they are toxic agents and are prevalent in the environment. Gold and platinum were of interest in this study since they increasingly are used as chemotherapeutic agents with recognized or possible toxic side effects. Sasaki and Watanabe ( 5 ) have previously attempted the analysis of unashed biological samples, but their study relied on high resolution to separate organic lines from elemental lines. In our study, organic interferences were held to lower molecular weight fragments by careful control of sparking parameters. It is through this method and the use of calibration curves that the direct analysis of biological materials for heavy metals appears to be a successful survey tool. EXPERIMENTAL A p p a r a t u s . T h e instrumen1.ation used in this study was an AEI MS-702 spark-source mass spectrometer which has been described elsewhere ( 1 2 ) ; with the only changes being made in the developer and developing conditions. These changes will be discussed later. R e a g e n t s . Ultra superior purity graphite (Ultra Carbon Corp., Bay City, Mich.) was used as matrix material for preparation of electrodes. Standard solutions of P t , Au. Hg. T1, P b , Bi, and U were prepared from platinum(ic) potassium chloride (Fisher), gold potassium cyanide (Fisher), mercuric oxide (Johnson M a t they). thallous nitrate (Fisher), lead oxide (Johnson Matthey), bismuth oxide (Johnson Matthey). and uranium oxide (Johnson Matthey) by dissolving the respective salt or oxide in electronic grade nitric or hydrochloric acid, followed by dilution t o give 1000-ppm stock solutions. All biological samples were obtained through the Department of Pathology. Blood serum from several donors was pooled in order to produce a representative medium blank. Homogenized liver samples (33% weight/volume j were obtained from control rats, and urine samples were obtained as needed from various donors. P r o c e d u r e . T h e best method of sample preparation was found t o be a lyophilization process to remove water, yielding residue in the form of a fine powder. Lyophilizing was accomplished with a commercial unit (Virtis) using a n alcohol-Dry Ice b a t h as a refrigerant. Serum samples mere prepared by adding 1.0 ml of serum to an acid-washed vial (2-dram) followed by t h e addition of 25 pg U internal standard. These samples were then spiked with known amounts of P t , Au. Hg. T1. P b , and Bi a t various concentrations a n d then lyophilized, yielding C Q . 80 mg of dry residue per 1.0 ml of wet serum. -411 metal additions were carried out with hlLA Precision Pipettes, 10-p1 a n d 25-p1, and were prepared directly from the shelf stock solutions or from diluted working stock solutions. Liver samples for analysis were prepared by adding 1.5 ml of the homogenized solution (ca. 0.5 gram of wet liver from the 33% homogenate) to an acid -washed glass vial (2-dram) followed by the addition of 25 pg U and the other elements of interest, followed by lyophilization. Urine samples required modification of procedure. Owing to the small solid content of urine coupled with sample adhesion to vessel surfaces, some problems were anticipated in the mechanical removal of the sample. For this reason, several sampling procedures were evaluated. T h e most successful method was use of a n ice shell between the walls of the glass vial and the lyophilizing urine sample. involving the formation of a thin layer of ice on the inside ot‘ the vial followed by the rapid addition and immediate freezing of the sample. T h e ice shell was obtained from 2.0 ml of distilled deionized water by swirling it in the vial (2-dram) with simultaneous cooling in liquid nitrogen. Once the shell was formed. the vial was kept in liquid nitrogen as 1.0 ml of spiked urine was added rapidly. The ice shell and urine were kept frozen until transfer to t h e lyophilizer. Lyophilization proceeded with little or no visible adhesion of the residue t o t h e glass walls. This lyophilate was very hygroscopic a n d required care in handling. Electrodes were prepared by mixing 20 mg of lyophilate with 100 mg of high purity graphite in a 10-ml acid-washed glass vial (with plastic snap cap) to which was added three small plastic (121 A W Fitchett and R P Buck. Ana/ Chem.. 45, 1027 (1973)
beads to aid in the homogenizing. Mixing was done for 15 minutes on a Spex mixer mill. A single electrode was formed using a polyethylene plug and a n AEI molding die. The electrode was broken in t h e middle and mounted in cleft indium tubes. The electrodes were sparked a t 40-kV spark voltage with the normal range of exposures being from 1 to 1000 nC with the pulse length a n d repetition rate changing accordingly to give adequate exposure rates. A 10-nC pre-sparking was made allowing the removal of small graphite specks which appeared as incandescent particles leaving the electrode surface. The AEI Autospark control unit was used in this study, not so much for the achievement of constant average spark gap, b u t more for the inherent convenience in making long exposures. After sparking, the photoplates were developed in a n internal image developer which will be discussed below.
RESULTS AND DISCUSSION Internal Standard. Uranium was chosen as the internal standard because it was a heavy element, essentially monoisotopic, and showed no interferences from organic molecular ions. Uranium was not expected to be present in these samples, although its presence cannot be totally ignored in samples of unknown history. Internal Image Developer. The original development process used for the photoplates paralleled that described previously (12). This produced plates with good contrast and low background fog. but the line intensities in the high mass range were not very strong. This made detection of the heavy elements difficult at low concentrations. Kennicott ( 1 3 , 1 4 ) and Cavard ( 1 5 ) have described developing reagents termed “internal developers” that yield the internal latent image rather than the surface latent image. Although described more than eight years ago, there does not appear t o be any widespread use of the process. Cavard’s internal developer. MK3, was compared to the standard surface .developer, Kodak D-19, in the following way. An electrode was prepared from a spiked serum sample and sparked in such a way that equal exposures were made on the top and bottom halves of a single photoplate. In the darkroom, the photoplate was carefully cut lengthwise to give two equal portions. One half was developed in D-19 under normal conditions and the other half developed in the internal developer for 4 minutes followed by normal stopping and fixing. By simultaneous evaluation of the same plate, it was possible to neglect any effects of possible emulsion changes from plate to plate. The results of this test were quite pronounced. All lines on the plate from the internal developer were much darker than the corresponding lines on the D-19 plate. Figure 1 shows a comparison of line intensities between the same lines for a given element for the two plates as measured from densitometer tracings. This figure indicates that much denser lines can be obtained a t shorter exposures or that much lower concentration levels can be seen at a given exposure level with the internal developer. The only problem encountered with the internal developer was increased background fog, but this was brought to a tolerable level by adjusting plate process times. Table I gives the revised plate development conditions which were used. No problems were encountered with degradation of the MK-3 developer with time or from thermoinstability. Sensitivity to temperature fluctuation during the development process was not observed because of precise control of the development process temperature. Sample-Graphite Ratios. In order to determine the proper sample/graphite ratio several electrode systems were (13) P R Kennicott. Thirteenth Ann. Conf. Mass Spectrom. Aliied Topics. 184 (1965) ( 1 4 ) P. R Kennicott. Anal. C h e m . 38. 6 3 3 (1966) ( 1 5 ) A Cavard. Adv Mass Spectrom.. 4, 419 (1968)
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~
Table I. Developing Conditions Developer
Internal developer, MK3 (15) for 4 minutes at
Stop bath
Eastman Kodak indicating stop bath (-14% acetic acid) for 30 seconds Eastman Kodak Rapid-Fixer with hardener for 2 minutes Running water for 7 minutes, distilled, deionized water for 1 minute Air drying at room temperature
18 "C
Fixer Washing Drying
Exposure,nC
Figure 1. Comparison between internal developer and Kodak D-19, see text l
o
r
08-
,
, ,
I
, ,
1
Serum
/
Figure 2. Calibration curve for serum; pg of impurity added to 1 .O mi serum 7
1 9 7 ~ " + ;
x 2 0 9 ~ ~ +0. 208pb+; A
density (at 1000 nC) vs.
203~1-
studied. Varying amounts of lyophilized sera spiked with 10 Fg each of Pt, Au, Hg, T1, Pb, and Bi were added to 100 mg
of graphite. The systems tried contained 80, 40, 20, 10, or 5 mg of lyophilate. These samples were mixed, formed into electrodes, and sparked. The ratio producing the most satisfactory spectra was 20 mg serum to 100 mg graphite, this ratio producing spectra with no organic interferences at the high mass range and showing adequate intensities of the added elements. Higher ratios showed some organic interferences in the region of interest; lower ratios, although demonstrating no organic background, gave very weak elemental lines. From this study, it was decided to prepare all samples from 20 mg of the lyophilate. Line Intensities. Line intensities were measured in the usual manner using a densitometer. In most cases the isotope with the highest abundance was used for the measurement with several exceptions. The largest problem encountered with the heavy metals arose from those possessing many isotopes. Platinum and mercury have six and seven isotopes, respectively, which tend to dilute the intensity of any given line, so that a t low concentrations the intensity of the most abundant isotope is still too weak for measurement, under conditions employed in this study. This suggests that platinum and mercury may be determined qualitatively from their distribution patterns but 712
ANALYTICAL CHEMISTRY, VOL. 46, NO. 6 , M A Y 1974
not quantitatively a t very low levels. The remaining elements, Au, T1, Pb, and Bi, were all measured with little difficulty. It should be noted that the weaker line of thallium, mass 203, was employed instead of the stronger line, mass 205, because of possible interference from the 13C containing polymer line adjacent to the C-17 a t mass 204. Sample Analysis. Serum samples were prepared by adding to 1.0-ml serum blanks the various trace elements in concentration ranges of 0.1 pg to 50 pg of each element along with 25 pg U internal standard. An attempt was made to determine relative sensitivity factors, but unlike previous study in this laboratory ( 2 2 ) , there was a strong dependence on concentration changes. The RSF values fluctuated with changes in concentration, thus rendering them useless. These changes may have been caused by fluctuations in electrode conditions and/or by the high organic content present. However, when a plot was made of the line density at the 1000-nC exposure level for each element us. the number of pg of that element added to serum, a linear response was obtained as depicted in Figure 2 (points represent an average of five samples). These data indicate that a fairly quantitative assay may be obtained for a serum sample from the graph directly by carrying out a 1000-nC exposure and relating line density observed to the element level present in the sample. The four elements shown are those measurable without interference. The use of calibration curve methods is discussed below. Liver samples were prepared and analyzed in the same manner as sera. As with serum, liver samples exhibited poor RSF correlation among samples of varying concentration. This may be ascribed to the fact that liver lyophilates do not furnish homogeneous matrices with the graphite; in preparation, the liver samples tended to be gum-like and in some cases formed a paste-like substance with the graphite. Despite this problem, sturdy electrodes with good sparking qualities were formed. Liver samples when plotted in the same way as the serum, demonstrated a linear response as shown in Figure 3 (each point representing an average of five samples at that concentration). Urine samples were prepared using an outer ice shell as mentioned above. Urine blank spectra were much cleaner than serum or tissue spectra relative to organic interferences, but tended to vary depending on the protein content. As a consequence. a urine specimen of high protein content was used for most studies, demonstrating the applicability of direct sampling. The cleaner spectra render available new mass regions of interest and, theoretically, a larger number of elements may be observed directly without organic interference. Many elements as far down as arsenic could be observed without organic interference including cadmium and antimony. A plot of added element (pg) us line density for a 300-nC exposure (not 1000 nC as with serum and liver) demonstrated a linear relationship as seen in Figure 4 (each point representing the average of five samples). With urine, it was observed that a 300-nC exposure was all that was needed for adequate line inten-
0
1
2
3
5
6
I
Figure 3. Calibration curve for homogenized liver; density (at 1000 nC) vs. pg of impurity added to 1.5 mi liver homogenate (33%) ‘(I 1 Y 7 A u + ; X 2 0 Y B i f ; 0 208pb+; A 2 0 3 T I +
sities due to the fact that a 20-mg sample of lyophilate represented almost the entire amount obtained from 1.0 ml. The use of calibration curves, as noted above, can be of considerable importance especially when employed in sample survey techniques. From the results presented, it can be seen that for a sample of unknown history a fairly accurate estimate of impurity concentration could be obtained from a single 1000-nC exposure for serum or liver, or a 300-nC exposure for urine. Once the concentration had been narrowed to a given range (flpg), a more precise single element technique such as atomic absorption could be employed. The use of a calibration curve method has been presented by Kai and Miki (16), but their system required the use of two standards to bracket the concentration of an unknown. All three samples were then shot on the same plate. The method presented here does not necessitate such a requirement. I t has been these authors’ experience that different Q2 photoplates from the same batch show nearly the same emulsion response, and, therefore, the use of one set of calibration curves for each photoplate batch is sufficient to obtain reliable quantitative results from one series of standards. It should also be noted that this (16) J K a i a n d M Miki M i t s u b s h i D e n k i Lab Rep 5 , 175 (1964)
1
2
3
4
5
6
Pg
Figure 4. Calibration curve for urine; density (at 300 nC) vs. wg of impurity added to 1 .O ml urine ‘(I 1 9 7 A u + ; X 2 o g B i + ; 0 2 0 8 P b + ; A 2 0 3 T I + ; 195pt+
method does not require the addition of an internal standard, although one was used, thus reducing the possibility of sample contamination. This study has attempted to demonstrate the feasibility of SSMS to the direct determination of heavy metals in serum, tissue, and urine. From Figures 2, 3, and 4, it can be seen that concentration levels down to fractions of a ppm can be realized, thus making this method a direct, low level survey technique for multielement analysis. By analyzing samples without chemical pretreatment or ashing, adulteration of the specimen is avoided; but the length of time required for lyophilizing can be a considerable drawback. Thus, a gain in purity is offset by increased preparation time. While time requirements and technique complexity are considerable a t this time, the procedure is a valuable multiple-element survey tool and should find application in regional surveys and other types of screening efforts. Received for review October 9, 1973. Accepted January 14, 1974. This paper was presented a t the 25th Southeastern Regional ACS Meeting, Charleston, South Carolina, November, 1973, paper No. 64. This work was supported by the University of Xorth Carolina Materials Research Center under Contract DAHC-15-67-CO223.
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